The primary goal of this project is to characterize the intracellular signal transduction mechanisms activated by fluid flow in endothelial cells (BC). This information will then be used to define the plasma membrane components by which endothelial cells sense their hemodynamic environment. This knowledge is important to our understanding of a wide variety of biological processes that are modulated by physical forces, such as bone growth, muscle hypertrophy, hair cell sound transduction, etc. The major hypothesis underlying the proposal is that fluid shear stress activates receptor-like signal events in BC. If this hypothesis is correct, it would follow that intracellular kinases (e.g., calcium- calmodulin dependent, protein kinase C (PKC), and a newly defined family termed mitogen activated protein kinases (MAPK)) should be activated by fluid shear stress. Our preliminary data provide strong support for a major role of the MAPK in the BC response to flow. To characterize the nature of the MAPK response to fluid shear stress and to identify other molecules that transmit the mechanical signal from plasma membrane to MAPK we will use immunological and biochemical techniques to study cultured human and bovine BC. These cells will be exposed to defined fluid shear stress in a parallel plate flow chamber. We will first characterize the MAPK response to flow by studying its dependence on shear stress, and its regulation by calcium, protein kinases (C kinase and tyrosine kinases), G proteins, and integrins. Next, we will study the differences in activation of MAPK by different flow patterns (steady, pulsatile and oscillatory). Finally, we will study the long-term alterations in these signal molecules in models (both in vivo and in vitro) of altered flow conditions. These studies should define the temporal and functional interactions of MAPK with other intracellular mediators activated by flow. Ultimately, the results will allow us to characterize and identify the plasma membrane components (i.e., the "shear stress receptor") responsible for the modulation of BC function by hemodynamic forces.